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of inhibitor of apoptosis protein (IAP) family members, Bax conformational changes .... PBS and analyzed with a fluorescence microscope (Carl Zeiss,. Germany).
INTERNATIONAL JOURNAL OF ONCOLOGY 42: 1036-1044, 2013

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Apoptosis induction of human prostate carcinoma cells by cordycepin through reactive oxygen species‑mediated mitochondrial death pathway HYE HYEON LEE1,2, CHEOL PARK3,4, JIN-WOO JEONG3,4, MIN JEONG KIM1,2, MIN JEONG SEO2, BYOUNG WON KANG2, JEONG UCK PARK2, GI-YOUNG KIM5, BYUNG TAE CHOI6, YUNG HYUN CHOI3,4,7 and YONG KEE JEONG1,2 1

Department of Biotechnology, 2Medi-Farm Industrialization Research Center, Dong-A University, Busan 604-714; 3 Department of Biochemistry, Dongeui University College of Oriental Medicine, Busan 614-052; 4 Anti‑Aging Research Center and Blue-Bio Industry RIC, Busan 614-714; 5Laboratory of Immunobiology, Department of Marine Life Sciences, Jeju National University, Jeju 690-756; 6Division of Meridian and Structural Medicine, School of Korean Medicine, Pusan National University, Gyeongnam 626-870; 7 Department of Biomaterial Control (BK21 program), Dongeui University, Busan 614-714, Republic of Korea Received October 22, 2012; Accepted December 7, 2012 DOI: 10.3892/ijo.2013.1762

Abstract. Cordycepin is the main functional component of Cordyceps militaris, which has been widely used in oriental traditional medicine. This compound has been shown to possess many pharmacological properties, such as enhancing the body's immune function, and anti-inflammatory, anti-aging and anticancer effects. In the present study, we investigated the apoptotic effects of cordycepin in human prostate carcinoma cells. We found that treatment with cordycepin significantly inhibited cell growth by inducing apoptosis in PC-3 cells. Apoptosis induction of PC-3 cells by cordycepin showed correlation with proteolytic activation of caspase-3 and -9, but not caspase-8, and concomitant degradation of poly (ADP-ribose) polymerases, collapse of the mitochondrial membrane potential (MMP). In addition, cordycepin treatment resulted in an increase of the Bax/Bcl-2 (or Bcl-xL) ratio, downregulation of inhibitor of apoptosis protein (IAP) family members, Bax conformational changes, and release of cytochrome c from the mitochondria to the cytosol. The cordycepin-induced apoptosis was also associated with the generation of intracellular reactive oxygen species (ROS). However, the quenching of ROS generation with antioxidant N-acetyl-L-cysteine conferred significant

Correspondence to: Dr Yong Kee Jeong, Department of Biotechnology, Dong-A University, Busan 604-714, Republic of Korea E-mail: [email protected]

Dr Yung Hyun Choi, Department of Biochemistry, Dongeui University College of Oriental Medicine, San 45, Yangjung-dong Busanjin-gu, Busan 614-052, Republic of Korea E-mail: [email protected]

Key words: cordycepin, PC-3 cells, apoptosis, reactive oxygen species

protection against cordycepin-elicited ROS generation, disruption of the MMP, modulation of Bcl-2 and IAP family proteins, caspase-3 and -9 activation and apoptosis. This indicates that the cellular ROS generation plays a pivotal role in the initiation of cordycepin-triggered apoptotic death. Collectively, our findings suggest that cordycepin is a potent inducer of apoptosis of prostate cancer cells via a mitochondrial-mediated intrinsic pathway and that this agent may be of value in the development of a potential therapeutic candidate for both the prevention and treatment of cancer. Introduction Prostate cancer is the most frequently diagnosed malignancy and the second most common cause of cancer death in men. It occurs predominantly in persons over 50 years of age (1), typically progressing at a slow rate (2). Prostate cancer in elderly males accounts for 33% of all newly diagnosed malignancies among men in the United States (3). Moreover, the number of patients with prostate cancer is increasing in Asia (4,5). Therefore, the exploration and development of novel and more effective antitumor agents for patients with prostate cancer are urgently needed. Apoptosis is a highly regulated process of programmed cell death that plays an important role in the maintenance of cellular homeostasis. Disruption of this process represents a major contributing factor in the pathology of cancer. Thus, apoptosis activation has been considered a good target in cancer therapies (6,7). In general, apoptosis is regulated by pro-apoptotic and anti-apoptotic gene products, such as the Bcl-2 and inhibitor of apoptosis protein (IAP) family members, and executed through caspases and cysteine-aspartic proteases, chiefly via two major and inter-related pathways (i.e., the mitochondriadependent ‘intrinsic’ cytochrome c/caspase-9 pathway and the death receptor-mediated ‘extrinsic’ caspase-8 pathway) (8,9).

LEE et al: INDUCTION OF APOPTOSIS BY CORDYCEPIN

Caspase activation further leads to protein cleavage resulting in DNA fragmentation, chromatin condensation and cell shrinkage. Additionally, reactive oxygen species (ROS) play a key role in mitochondria-mediated apoptosis. Mitochondria are the prime source of ROS, which are byproducts of aerobic respiration (10,11). High levels of ROS in mitochondria can result in free radical attack of membrane phospholipids and cause mitochondrial membrane depolarization. This is an irreversible step, which is associated with the release of mitochondrial factors including cytochrome c, triggering caspase cascades (12,13,14). Therefore, ROS plays an important role in mitochondria-mediated apoptotic pathway. Cordycepin, 3'-deoxyadenosine, is a major functional component in the Cordyceps militaris fungus (Fig. 1) (15,16). Due to the absence of oxygen in the 30-position of its ribose moiety, the incorporation of cordycepin during RNA synthesis will result in termination of chain elongation. This activity has been well described in vitro with purified RNA polymerases and poly(A) polymerases from a number of organisms, including yeast and mammals (17,18). Cordycepin has also demonstrated various properties, such as antitumor (18,19,20), anti‑fungal (21), anti‑bacterial (22) and anti‑inflammatory effects (23,24). Indeed, for centuries, Cordyceps militaris has been a widely administered traditional Chinese medicine, with cordycepin believed to be one of the bioactive components mediating its beneficial effects (16,25). While well known as a therapeutic agent due to its unique properties, the molecular mechanisms underlying the anticancer effects of cordycepin are not yet completely understood. The purpose of this study was to evaluate the role of mitochondria in apoptosis induced by cordycepin, using human prostate carcinoma cells. We examined whether ROS were critical mediators of cordycepin-induced PC-3 cell death, and we determined the sequence of events leading to the activation of downstream caspases and apoptosis. The study furnishes evidence that cordycepin elicits ROS, which in turn triggers a decrease in mitochondria membrane potential (MMP), consequently leading to caspase activation. Materials and methods Reagents and antibodies. Cordycepin (MW, 251.2; product no. C3394), 4,6-diamidino-2-phenylindole (DAPI), dimethyl sulfoxide (DMSO), N-acetyl-L-cysteine (NAC), 3-(4,5-dimethyl2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT), 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethyl-imidacarbocyanine iodide (JC-1) and propidium iodide (PI) were purchased from the Sigma‑Aldrich Chemical Co. (St. Louis, MO). Fetal bovine serum (FBS) and caspase activity assay kits were obtained from Gibco-BRL (Grand Island, NY) and R&D Systems (Minneapolis, MN), respectively. The DNA staining kit (CycleTEST™ Plus Kit) and enhanced chemiluminescence (ECL) kit were purchased from Becton‑Dickinson (San Jose, CA) and Amersham Co. (Arlington Heights, IL), respectively. Antibodies specific for XIAP, cIAP-1, cIAP-2, Bcl-2, Bax, Bcl-xL, caspase-3, -8 and -9, and poly(ADP‑ribose)polymerases (PARP) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-cytochrome c and actin antibodies were purchased from Cell Signaling (Beverly, MA) and Sigma-Aldrich Chemical Co., respectively. The peroxidase-labeled donkey anti-rabbit immunoglobulin and

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peroxidase-labeled sheep anti-mouse immunoglobulin were purchased from Amersham Co. Cell lines, cell culture and MTT assay. Human prostate cancer cell lines (PC-3, DU145 and LNCaP) were obtained from the American Type Culture Collection (Rockville, MD). The culture medium used throughout the experiments was RPMI‑1640 medium (Gibco-BRL), containing 10% FBS, 2 mM L-glutamine, and 100 U/ml penicillin and streptomycin. Cells were cultured at 37˚C in a humidified chamber containing 5% CO2. For the cell viability assay, cells were seeded in 6-well plates and treated with various concentrations of cordycepin for 24 h. After treatments, MTT working solution was added to 6-well culture plates and incubated continuously at 37˚C for 2 h. The culture supernatant was removed from the wells and DMSO was added to dissolve the formazan crystals. The absorbance of each well was measured at 540 nm with an ELISA reader (Molecular Devices, Sunnyvale, CA). Flow cytometry analysis. After treatment with cordycepin, the cells were collected, washed with cold phosphate-buffered saline (PBS) and fixed in 75%  ethanol at 4˚C for 30  min. Prior to analysis, the cells were washed once again with PBS, suspended in a cold PI solution containing 100 µg/ml RNase A, 50 µg/ml PI, 0.1% (w/v) sodium citrate and 0.1% (v/v) NP-40, and further incubated on ice for 30 min in the dark. Flow cytometry analyses were carried out using a flow cytometer (FACSCalibur; Becton‑Dickinson). Cell-Quest software was used to determine the relative DNA content based on the presence of red fluorescence. The sub-G1 population was calculated to estimate the apoptotic cell population (26). DNA fragmentation assay. Cells were lysed in 100 µl of 10 mM Tris‑HCl buffer (pH 7.4) containing 10 mM EDTA and 0.5% Triton X‑100. After centrifugation for 5 min at 15,000 rpm, supernatant samples were treated with RNase A and proteinase K. Subsequently, 20 µl of 5 M NaCl and 120 µl isopropanol were added to the samples, which were then kept at -20˚C for 6 h. Then, following centrifugation for 15 min at 15,000 rpm, DNA pellets were dissolved in 20 µl of TE buffer (10 mM Tris‑HCl and 1 mM EDTA) as loading samples. To assay the DNA fragmentation pattern, samples were loaded onto 1.5% agarose gel and electrophoresis was carried out. DAPI staining. Cells were washed with cold PBS and fixed with 4% paraformaldehyde (Sigma-Aldrich Chemical Co.) in PBS for 10 min at room temperature. The fixed cells were washed with PBS and stained with DAPI solution for 10 min at room temperature. The cells were then washed twice with PBS and analyzed with a fluorescence microscope (Carl Zeiss, Germany). Determination of caspase activity. The activities of caspases were determined by colorimetric assay kits, which utilize synthetic tetrapeptides [Asp-Glu-Val-Asp (DEAD) for caspase-3; Ile-Glu-Thr-Asp (IETD) for caspase-8; and Leu-Glu-His-Asp (LEHD) for caspase-9] labeled with p‑nitroaniline (pNA), according to the manufacturer's protocol. The cells were briefly lysed in the supplied lysis buffer. The supernatants were collected and incubated with

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INTERNATIONAL JOURNAL OF ONCOLOGY 42: 1036-1044, 2013

Figure 1. The chemical structure of cordycepin.

the supplied reaction buffer containing dithiothreitol (DTT) and substrates at 37˚C for 2 h in the dark. The caspase activity was determined by measuring changes in absorbance at 405 nm using the ELISA reader. Isolation of total‑RNA and reverse transcription-PCR. Total‑RNA was isolated using TRIzol reagent (Invitrogen, Carlsbad, CA). Total‑RNA (1.0 µg) obtained from cells was primed with random hexamers to synthesize complementary DNA using M-MLV reverse transcriptase (Promega, Madison, WI) according to the manufacturer's instructions. Single stranded cDNA was amplified by polymerase chain reaction (PCR) with the indicated primers. Amplification products obtained by PCR were electrophoretically separated on 1% agarose gel and visualized by ethidium bromide (EtBr, Sigma-Aldrich Chemical Co.) staining. Protein extraction and western blot analysis. The cells were harvested and lysed with lysis buffer (20 mM sucrose, 1 mM EDTA, 20 µM Tris‑Cl, pH 7.2, 1 mM DTT, 10 mM KCl, 1.5 mM MgCl2 and 5 µg/ml aprotinin) for 30 min. The protein concentration was measured using a Bio-Rad protein assay (Bio-Rad Laboratories, Hercules, CA) according to the manufacturer's instructions. In a parallel experiment, cells were washed with cold PBS and scraped; cytoplasmic and nuclear proteins were then extracted using a mitochondrial fractionation kit according to the manufacturer's instructions (Activemotif, Carlsbad, CA). For western blot analysis, an equal amount of protein was subjected to electrophoresis on SDS-polyacrylamide gel and transferred by electroblotting to a nitrocellulose membrane (Schleicher & Schuell, Keene, NH). The blots were probed with the desired antibodies for 1 h, incubated with the diluted enzyme-linked secondary antibody and visualized by ECL kit according to the recommended procedure. Mitochondrial membrane potential (MMP, ∆Ψm) assay. The MMP of intact cells was measured by DNA flow cytometry with the lipophilic cation JC-1. JC-1 is a ratiometric, dual‑emission fluorescent dye that is internalized and concentrated by respiring mitochondria; therefore, it can reflect changes in MMP in living cells. There are two excitation wavelengths: at low values of MMP, it remains a monomer (FL-1, green fluorescence; 527 nm) while it forms aggregates at high MMP (FL-2, red fluorescence; 590 nm), according to the recommended procedure (Calbiochem). For this study, the cells were trypsinized and the cell pellets were resuspended in PBS and incubated with 10 µM JC-1 for 20 min at 37˚C. The cells were subsequently washed once with cold PBS, suspended and analyzed using a flow cytometer.

Measurement of intracellular ROS generation. The generation of ROS was determined in cells treated with cordycepin in the presence and absence of NAC, and was evaluated with 5-(and 6)-carboxy-2'7'-dichlorodihydrofluorescein diacetate (DCF‑DA; Molecular Probes, Leiden, The Netherlands) as described previously (27). The cells were incubated with 10 µM DCF-DA at 37˚C for 30 min. The cells were then washed with PBS and FL-1 fluorescence was measured with a flow cytometer. Statistical analysis. The data are expressed as a mean ± SD. A statistical comparison was performed using one-way ANOVA followed by a Fisher's test. The significant (p